Target (protein/gene name): UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diaminopi melate-D-alanyl-D-alanyl ligase (Rv2158c) NCBI Gene # or RefSeq#: NC_000962.3 Protein ID (NP or XP #) or Wolbachia#: NP_216674.1 Organism (including strain): Mycobacterium tuberculosis (H37Rv) Etiologic Risk Group (see link below): N/A Disease Information (sort of like the Intro to your Mini Research Write up): Mycobacterium tuberculosis is a pathogenic-type bacteria that is in charge of the infectious Tuberculosis disease. Tuberculosis is highly infective, known for having a high morbidity and a high mortality rate. The disease starting infecting more and becoming more severe after 1985 when HIV entered into body systems and lowered the immunity of the infected. The severity of the bacteria is contagious by its microscopic bodily fluids out in the air that can infect the surface in the lungs which secretes pulmonary surfactant that decreases the surface tension of the alveoli. The most common symptoms in a Tuberculosis-infected patient are coughing, fatigue, a high fever, and chest pain. Those who have poor nutritional diets, live in places that do not offer medical care, or are highly populated risk getting Tuberculosis. Because of its resiliency tendency, M.tuberculosis bacterium undergo quick mutations resulting in more resistant strains compared to other bacteria, so it is important to keep up with the strains and research in order to prevent the spread of the disease.
Current Inhibitors: Nine compounds for the Target in M. tuberculosis
Expression Information (has it been expressed in bacterial cells):
[1] Chandrakala Basavannacharya, Giles Robertson, Tulika Munshi, Nicholas H. Keep, Sanjib Bhakta Tuberculosis (Edinb) 2010 Jan; 90(1): 16–24. Published online 2009 Nov 27. doi: 10.1016/j.tube.2009.10.007 ATP-dependent MurE ligase in Mycobacterium tuberculosis: biochemical and structural characterisation. [2] Basavannacharya C, Moody PR, Munshi T, Cronin N, Keep NH, Bhakta S. Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis. Protein & Cell. 2010;1(11):1011-1022. doi:10.1007/s13238-010-0132-9.
Image of protein (PyMol with features delineated and shown separately): Fig 1. Crystal structure of Mycobacterium tuberculosis UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diaminopi melate-D-alanyl-D-alanyl ligase compared with the 2WTZ MurE ligase
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
>2WTZ:A|PDBID|CHAIN|SEQUENCE
MSSLARGISRRRTEVATQVEAAPTGLRPNAVVGVRLAALADQVGAALAEGPAQRAVTEDRTVTGVTLRAQDVSPGDLFAA
LTGSTTHGARHVGDAIARGAVAVLTDPAGVAEIAGRAAVPVLVHPAPRGVLGGLAATVYGHPSERLTVIGITGTSGKTTT
TYLVEAGLRAAGRVAGLIGTIGIRVGGADLPSALTTPEAPTLQAMLAAMVERGVDTVVMEVSSHALALGRVDGTRFAVGA
FTNLSRDHLDFHPSMADYFEAKASLFDPDSALRARTAVVCIDDDAGRAMAARAADAITVSAADRPAHWRATDVAPTDAGG
QQFTAIDPAGVGHHIGIRLPGRYNVANCLVALAILDTVGVSPEQAVPGLREIRVPGRLEQIDRGQGFLALVDYAHKPEAL
RSVLTTLAHPDRRLAVVFGAGGDRDPGKRAPMGRIAAQLADLVVVTDDNPRDEDPTAIRREILAGAAEVGGDAQVVEIAD
RRDAIRHAVAWARPGDVVLIAGKGHETGQRGGGRVRPFDDRVELAAALEALERRA
length of your protein in Amino Acids
535 AA Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website
55.34076 kDa Molar Extinction efficient of your protein at 280 nm wavelenth:
18575
TMpred graph Image TMPRED
Input your amino acid sequence to it.
Fig. 2 TMpred output for UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diaminopi melate-D-alanyl-D-alanyl ligase
Do Not Need this info for Spring (but still copy these lines to your Target page for now
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers
link to DNA Works output m9l40l772 that should be saved in your folder after you did the primer design protocol
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not< sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
NCBI Gene # or RefSeq#: NC_000962.3
Protein ID (NP or XP #) or Wolbachia#: NP_216674.1
Organism (including strain): Mycobacterium tuberculosis (H37Rv)
Etiologic Risk Group (see link below): N/A
Disease Information (sort of like the Intro to your Mini Research Write up): Mycobacterium tuberculosis is a pathogenic-type bacteria that is in charge of the infectious Tuberculosis disease. Tuberculosis is highly infective, known for having a high morbidity and a high mortality rate. The disease starting infecting more and becoming more severe after 1985 when HIV entered into body systems and lowered the immunity of the infected. The severity of the bacteria is contagious by its microscopic bodily fluids
out in the air that can infect the surface in the lungs which secretes pulmonary surfactant that decreases the surface tension of the alveoli. The most common symptoms in a Tuberculosis-infected patient are coughing, fatigue, a high fever, and chest pain. Those who have poor nutritional diets, live in places that do not offer medical care, or are highly populated risk getting Tuberculosis. Because of its resiliency tendency, M.tuberculosis bacterium undergo quick mutations resulting in more resistant strains compared to other bacteria, so it is important to keep up with the strains and research in order to prevent the spread of the disease.
Link to TDR Targets page (if present):
http://www.tdrtargets.org/targets/view?gene_id=6551
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
https://www.patricbrc.org/view/Feature/PATRIC.1643430.3.LDXW01000001.CDS.103333.104823.rev
https://www.ncbi.nlm.nih.gov/gene/?term=2.4.2.8+mycobacterium+tuberculosis
Essentiality of this protein:
Rv2158c has essentiality data
Gene/Ortholog: eco86 (OG4_14754); Phenotype: undefined; Source study: blattner
Gene/Ortholog: eco86 (OG4_14754); Phenotype: essential; Source study: gerdes
Gene/Ortholog: eco86 (OG4_14754); Phenotype: essential; Source study: keio
Gene/Ortholog: eco86 (OG4_14754); Phenotype: essential; Source study: shigen
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
No it is not.
EC#:
**6.3.2.13**
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=6.3.2.13&UniProtAcc=P9WJL3&OrganismID=3445
Link to BRENDA HPTR Pathway:
http://www.brenda-enzymes.org/pathway_index.php?menu=off&site=pathway&search_mode=exact&pathway=peptidoglycan_biosynthesis&ecno=6.3.2.13
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
colorimetric phosphate detection method
[1] https://www.researchgate.net/publication/45707755_Anti-tubercular_screening_of_natural_product_from_Colombian_plants_3-methoxynordomesticine_an_inhibitor_of_MurE_ligase_of_Mycobacterium_tuberculosis
[2] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498294/
Fluorogenic substrates, DMSO and PBS
DMSO - $68.50 for 100 mL
PBS - $110 for 1L
Structure (PDB or Homology model)
PDB # or closest PDB entry if using homology model:
2WTZ
http://www.rcsb.org/structure/2WTZ
Current Inhibitors:
Nine compounds for the Target in M. tuberculosis
Expression Information (has it been expressed in bacterial cells):
[1] Chandrakala Basavannacharya, Giles Robertson, Tulika Munshi, Nicholas H. Keep, Sanjib Bhakta Tuberculosis (Edinb) 2010 Jan; 90(1): 16–24. Published online 2009 Nov 27. doi: 10.1016/j.tube.2009.10.007 ATP-dependent MurE ligase in Mycobacterium tuberculosis: biochemical and structural characterisation.
[2] Basavannacharya C, Moody PR, Munshi T, Cronin N, Keep NH, Bhakta S. Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis. Protein & Cell. 2010;1(11):1011-1022. doi:10.1007/s13238-010-0132-9.
Image of protein (PyMol with features delineated and shown separately):
Fig 1. Crystal structure of Mycobacterium tuberculosis UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diaminopi melate-D-alanyl-D-alanyl ligase compared with the 2WTZ MurE ligase
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
>2WTZ:A|PDBID|CHAIN|SEQUENCE
MSSLARGISRRRTEVATQVEAAPTGLRPNAVVGVRLAALADQVGAALAEGPAQRAVTEDRTVTGVTLRAQDVSPGDLFAA
LTGSTTHGARHVGDAIARGAVAVLTDPAGVAEIAGRAAVPVLVHPAPRGVLGGLAATVYGHPSERLTVIGITGTSGKTTT
TYLVEAGLRAAGRVAGLIGTIGIRVGGADLPSALTTPEAPTLQAMLAAMVERGVDTVVMEVSSHALALGRVDGTRFAVGA
FTNLSRDHLDFHPSMADYFEAKASLFDPDSALRARTAVVCIDDDAGRAMAARAADAITVSAADRPAHWRATDVAPTDAGG
QQFTAIDPAGVGHHIGIRLPGRYNVANCLVALAILDTVGVSPEQAVPGLREIRVPGRLEQIDRGQGFLALVDYAHKPEAL
RSVLTTLAHPDRRLAVVFGAGGDRDPGKRAPMGRIAAQLADLVVVTDDNPRDEDPTAIRREILAGAAEVGGDAQVVEIAD
RRDAIRHAVAWARPGDVVLIAGKGHETGQRGGGRVRPFDDRVELAAALEALERRA
length of your protein in Amino Acids
535 AA
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website
55.34076 kDa
Molar Extinction efficient of your protein at 280 nm wavelenth:
18575
TMpred graph Image TMPRED
Input your amino acid sequence to it.
Fig. 2 TMpred output for UDP-N-acetylmuramoylalanyl-D-glutamyl-2, 6-diaminopi melate-D-alanyl-D-alanyl ligase
**Protein Sequence (paste as text only):** >>Mycobacterium tuberculosis H37Rv|Rv2158c|murE 10 20 30 40 50 MSSLARGISR RRTEVATQVE AAPTGLRPNA VVGVRLAALA DQVGAALAEG 60 70 80 90 100 PAQRAVTEDR TVTGVTLRAQ DVSPGDLFAA LTGSTTHGAR HVGDAIARGA 110 120 130 140 150 VAVLTDPAGV AEIAGRAAVP VLVHPAPRGV LGGLAATVYG HPSERLTVIG 160 170 180 190 200 ITGTSGKTTT TYLVEAGLRA AGRVAGLIGT IGIRVGGADL PSALTTPEAP 210 220 230 240 250 TLQAMLAAMV ERGVDTVVME VSSHALALGR VDGTRFAVGA FTNLSRDHLD 260 270 280 290 300 FHPSMADYFE AKASLFDPDS ALRARTAVVC IDDDAGRAMA ARAADAITVS 310 320 330 340 350 AADRPAHWRA TDVAPTDAGG QQFTAIDPAG VGHHIGIRLP GRYNVANCLV 360 370 380 390 400 ALAILDTVGV SPEQAVPGLR EIRVPGRLEQ IDRGQGFLAL VDYAHKPEAL 410 420 430 440 450 RSVLTTLAHP DRRLAVVFGA GGDRDPGKRA PMGRIAAQLA DLVVVTDDNP 460 470 480 490 500 RDEDPTAIRR EILAGAAEVG GDAQVVEIAD RRDAIRHAVA WARPGDVVLI 510 520 530 AGKGHETGQR GGGRVRPFDD RVELAAALEA LERRA